During development, angiogenesis mediated by vascular endothelial growth factor (VEGF) signaling is extremely active in the central nervous system (CNS). The newly formed vessel is often immature and sensitive to bleeding and thus requires special mechanisms to maintain its endothelial barrier stability. However, known mechanisms for vascular integrity, such as blood-brain-barrier function, are not fully developed before birth. Additional mechanisms for vascular stability during CNS development remain elusive. We have recently identified a novel mechanism of platelet activation in which the O-glycoprotein podoplanin (PDPN) activates platelet C-type lectin-like 2 (CLEC-2) receptors. Our preliminary experiments show that PDPN is expressed specifically in neural cells surrounding vessels in the early developing CNS. Mice lacking PDPN or CLEC-2 (Pdpn-/- or Clec-2-/-) develop CNS-specific hemorrhages primarily during early embryonic development. Furthermore, mice with conditional deletion of PDPN in neural cells also exhibit spontaneous brain bleeding. These preliminary results support a novel hypothesis that neural cell PDPN-mediated platelet activation is essential for the stability of nascent vessels in the early developing CNS. To test this hypothesis, we will determine 1) whether PDPN-CLEC-2-mediated platelet activation protects integrity of newly formed vessels in the early developing CNS. We found that PDPN is expressed on neural cells closely associated with vessels in the early developing CNS and that platelets are present outside vessels. These data support a novel hypothesis that interactions between PDPN on perivascular neural cells and CLEC-2 on extravasated platelets are essential for vascular integrity of newly formed vessel during early CNS development. We will test this using the state-of-the-art two-photon confocal imaging microscopy and vascular permeability assays; we will determine 2) whether sphingoshine 1-phosphate (S1P) released from PDPN-CLEC-2-activated platelets balances VEGF action to maintain vascular stability in the developing CNS. We hypothesize that release of S1P from platelets after PDPN-CLEC-2-mediated activation is essential for vascular endothelial barrier stability by balancing the functin of VEGF, and will use mice lacking S1P functions and pharmacological approaches to address this question. If the proposed studies support our hypotheses, it will define a novel mechanism of tissue-specific platelet activation in regulation of vascular integrity in the developing CNS. Identification of such a new mechanism may provide new insights into brain bleeding disease, such as germinal matrix- intraventricular hemorrhage (GMH-IVH), which affects ~35% of premature human infants. In addition, Pdpn-/- or Clec-2-/- mice may be used as a valuable model for testing novel therapies promoting vascular integrity that target hemorrhage in the developing brain.